Skeletal muscle adapts to endurance exercise with increases in mitochondria and GLUT4. The goal of this research is to discover the mechanisms by which exercise induces these adaptations. We have found that intermittently increasing cytosolic Ca 2. in muscle cells stimulates mitochondrial biogenesis and GLUT4 expression. This effect of Ca2. is mediated by activation of Ca2*-calmodulin dependent protein kinases (CAMKs). We have also shown that activation of AMP-activated protein kinase (AMPK) has a similar effect. The transcription factors NRF-1 and NRF-2 activate nuclear genes that encode a number of mitochondrial respiratory chain proteins as well as mitochondrial transcription factor A mt(TFA), which activates mitochondrial DNA transcription and replication. PPAR7 coactivator lcx (PGC-I_) coactivates NRF-1 and PPARcx, and induces increases in expression of NRF-1 and NRF-2. Overexpression of PGC-lc_ in myocytes greatly stimulates mitochondrial biogenesis. We have shown that a bout of exercise induces increases in PGC-I_ protein expression and also of NRF-1 and NRF-2. We have also found that activation of CAMKs by raising cytosolic Ca 2., and of AMPK with AICAR induces rapid increases in PGC-lc_, NRF-1, NRF-2 and mt(TFA) in L6 myotubes. One of our goals is to identify the steps leading from activation of CAMK and AMPK to increased mitochondrial biogenesis and GLUT4 expression. Relative to this goal our aims are to test the hypotheses that activation of p38 MAPK is the next step in the pathway leading from CAMK and AMPK to increased mitochondrial biogenesis and GLUT expression, that nitric oxide synthase is involved in this process, and that activation of p38 results in phosphorylation and activation of PGC-I_. A second goal is to determine whether phosphorylation and activation of PGC-lcc initiates the stimulation of mitochondrial biogenesis and GLUT4 expression, and that this process is then amplified and maintained by an increase in PGC-I(x protein. A third goal is to determine whether the initial, very rapid, increase in some mitochondrial proteins is mediated by increased translation of preexisting mRNAs. A fourth goal is to elucidate the role of calcineurin in the stimulation of mitochondrial biogenesis by exercise and by raising cytosolic Ca 2.. A fifth goal is to elucidate the role of free fatty acids in the stimulation of mitochondrial biogenesis, to determine whether or not their effect is limited to induction of increases in the fatty acid oxidation pathway enzymes, and to elucidate their mechanisms of action. Our sixth goal is to evaluate the possibility that treatment with metformin will stimulate mitochondrial biogenesis and GLUT4 expression in muscle. It is our hope that information obtained from this research may make it possible to mimic some of the beneficial effects of exercise by pharmacological or gene therapy interventions in individuals who are unable to exercise.